Glycyrrhizin Binds to High-Mobility Group Box 1 Protein and Inhibits Its Cytokine Activities

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Glycyrrhizin Binds to High-Mobility Group Box 1 Protein and Inhibits Its Cytokine Activities
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  Chemistry & Biology  Article Glycyrrhizin Binds to High-Mobility Group Box 1Protein and Inhibits Its Cytokine Activities Luca Mollica, 1 Francesco De Marchis, 2  Andrea Spitaleri, 1 Corrado Dallacosta, 1 Danilo Pennacchini, 1 Moreno Zamai, 3  Alessandra Agresti, 2 Lisa Trisciuoglio, 2 Giovanna Musco, 1,5, * and Marco E. Bianchi 2,4,5, * 1 Biomolecular NMR Laboratory, Dulbecco Telethon Institute 2 Chromatin Dynamics 3 Dynamic Fluorescence Spectroscopy in BiomedicineSan Raffaele Scientific Institute, via Olgettina 58, 20133 Milan, Italy 4 Faculty of Medicine, San Raffaele University, via Olgettina 58, 20133 Milan, Italy 5 These authors contributed equally to this work.*Correspondence: musco.giovanna@hsr.it (G.M.), bianchi.marco@hsr.it (M.E.B.) DOI 10.1016/j.chembiol.2007.03.007 SUMMARY  High-mobility group box 1 protein (HMGB1) is anuclearcomponent, but extracellularly it servesas a signaling molecule involved in acute andchronic inflammation, for example in sepsisandarthritis.TheidentificationofHMGB1inhib-itorsisthereforeofsignificantexperimentalandclinical interest. We show that glycyrrhizin, anatural anti-inflammatory and antiviral triter-pene in clinical use, inhibits HMGB1 chemo-attractant and mitogenic activities, and has aweak inhibitory effect on its intranuclear DNA-binding function. NMR and fluorescence stud-ies indicate that glycyrrhizin binds directly toHMGB1 (K  d   150  m M), interacting with twoshallow concave surfaces formed by the twoarms of both HMG boxes. Our results explainin part the anti-inflammatory properties of gly-cyrrhizin, and might direct the design of newderivatives with improved HMGB1-bindingproperties. INTRODUCTION Inflammation, in the broadest sense, is a physiological,protectiveresponsetoinjuryandinfection.Acomplexnet-work of cellular responses leads to the resolution of infec-tionand/ortherepairofthedamagedtissue.Alterationsof this process, however, are at the basis of many acute anddegenerative diseases, including, among others, sepsis,atherosclerosis, and arthritis.High-mobility group box 1 (HMGB1) protein is a nuclearprotein that acts as an architectural chromatin-bindingfactor [1, 2]. HMGB1 was identified in 1999 as an impor-tant extracellular mediator of inflammation [3]. When cellsdie in a nonprogrammed way (necrosis), they releaseHMGB1 by simple diffusion; in contrast, cells that die ina programmed way (apoptosis) avidly retain HMGB1boundtochromatinremnantsevenaftereventualcelllysis[4]. This differential behavior between necrotic and apo-ptotic cells makes HMGB1 the primary signal of tissuedamage; extracellular HMGB1 promotes local and sys-temic responses in the organism, including inflammationand the activation of innate and adaptive immunity [5–7]. Activatedmonocytes,macrophages,neutrophils,plate-lets, and dendritic and NK cells can also release HMGB1in the extracellular space [3, 8–10]. These cells do notdie, but actively secrete HMGB1 in a process that is inde-pendent of the endoplasmic reticulum and the Golgiapparatus and depends on HMGB1 relocalization fromthenucleustospecialorganelles,thesecretorylysosomes[11–13].Extracellular HMGB1 activates a large number of differ-entphysiologicalresponsesindifferentcelltypes,andcanbe considered a cytokine [14]. Its beneficial roles includethe promotion of tissue regeneration, by attracting stemcells and inducing them to proliferate [15, 16]. However,HMGB1 plays a pathogenetic role in severe sepsis andarthritis [17, 18], and may play a role in atherosclerosisand cancer [19, 20]. Antibodies against HMGB1 can re-verse sepsis caused by peritonitis in mice [21]; therefore,the identification of small-molecule inhibitors of HMGB1might have significant therapeutic importance. A large number of anti-inflammatory drugs exist, andthe mode of action of several of these is unknown. AsHMGB1 wasrecognized as animportant proinflammatorymolecule only fairly recently, it looked likely that one ormore of the clinically effective anti-inflammatory drugscould exert their action by interfering with this relativelyunexplored inflammatory axis. Our attention focusedon glycyrrhizin, a glycoconjugated triterpene ( Figure 1 A)produced by the licorice plant,  Glycyrrhiza glabra . InJapan, glycyrrhizin is administered at high doses (up to140 mg/day) to patients with hepatitis B and C [22].Remarkably, our own work has highlighted a possiblerole of HMGB1 in the pathogenesis of hepatitis [23]. Theconvergence of these pieces of information identifiedglycyrrhizinasacandidateinhibitoroftheHMGB1-depen-dent inflammatory axis.We show here that indeed glycyrrhizin inhibits thechemoattractant and mitogenic activities of HMGB1. Chemistry & Biology  14 , 431–441, April 2007 ª 2007 Elsevier Ltd All rights reserved  431  Glycyrrhizin binds directly to each of the two HMG boxesof HMGB1, as shown by NMR and fluorescence studies. RESULTS Glycyrrhizin Inhibits the Chemoattractant Activity of HMGB1 HMGB1 has chemoattractant activity on endothelial,smooth muscle, and vessel-associated stem cells [15,24]. In addition, we found that it is also a powerful chemo-attractant for the widely available mouse 3T3 fibroblasts( Figure 1B). Glycyrrhizin interfered with HMGB1-elicitedcell migration of 3T3 fibroblasts in a dose-dependentmanner ( Figure 1B; p < 0.001 in ANOVA two-way test).The number of 3T3 cells migrating in response to 1 nMHMGB1 was reduced by about half at a concentration of 50  m M glycyrrhizin (IC 50  = 49 ± 1  m M in a sigmoidal logdoseresponsecurve,r 2 =0.93).Glycyrrhizindidnotaffectcell migration elicited by fMLP, a well-known chemo-attractant, even at a concentration of 200  m M. This showsthat glycyrrhizin does not affect the general mobility of fibroblasts, and suggests that its effect is specific onHMGB1-elicited chemotaxis. Moreover, glycyrrhizin hadno statistically significant effect on the mobility of fibro-blasts in the absence of chemoattractant ( Figure 1B). GlycyrrhizinInhibitstheMitogenicActivityofHMGB1on Vessel-Associated Stem Cells HMGB1 promotes the in vitro proliferation of vessel-associated stem cells (mesoangioblasts) in the absenceof fetal calf serum [15]. When added to mesoangioblastsin culture, increasing concentrations of glycyrrhizin Figure 1. Glycyrrhizin Inhibits the Chemotactic Activity of HMGB1 (A)Thechemicalstructureofglycyrrhizin.Carbenoxoloneisaderivativewherethetwoglucuronicacidresiduesaresubstitutedbyasuccinylresidue.(B) 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers where 0.1 nM fMLP, 1 nM HMGB1, or no chemoattractant was addedin the lower chamber, together with the indicated concentrations of glycyrrhizin. The data represent the average ± SD of three replicates; theexperiments were replicated at least three times. The inhibitory effects of glycyrrhizin on HMGB1-induced cell migration were highly significant(p < 0.001 in ANOVA analysis), whereas they were not statistically significant on fMLP-induced cell migration. Chemistry & Biology Glycyrrhizin Inhibits HMGB1 Activities 432  Chemistry & Biology  14 , 431–441, April 2007 ª 2007 Elsevier Ltd All rights reserved  inhibited the mitogenic activity of HMGB1 in a dose-response manner; in the presence of 200  m M glycyrrhizinno mitogenic activity of HMGB1 was noted, and the cellcounts were indistinguishable from those of cultureswhere no serum and no HMGB1 were present ( Figure 2 ).Conversely, glycyrrhizin showed no effect on growth of mesoangioblasts in the presence of 20% fetal calf serum(data not shown). Glycyrrhizin Binds Directly to HMGB1: Identificationof the Binding Surface by NMR Binding of glycyrrhizin to HMGB1 was probed using NMRchemical-shiftdifferences(CSD),ahighlysensitivetoolforprovinginteractionsandformappingbindingsitesandde-tecting residues which directly interact with the ligand orthatareindirectlyaffectedbytheassociation.Two-dimen-sional  1 H- 15 N HSQC spectra of full-length  15 N-labeledHMGB1 were recorded to monitor the changes in the 1 H- 15 N chemical shifts of the backbone amide groups in-duced by successive additions of glycyrrhizin. A compar-isonofthespectraintheabsenceandpresenceofa4-foldexcess of glycyrrhizin is shown in Figure 3 A. The complexis in fast exchange on the chemical-shift timescale( Figure 3B); this facilitated the assignments of the reso-nances in the complex, which were obtained followingthe crosspeaks of HMGB1 upon addition of increasingamounts of glycyrrhizin.To identify the interaction surface, the average chemi-cal-shift changes between the free and the bound statewere plotted versus the HMGB1 residue numbers ( Fig-ure 3C). Most of the protein residues do not experiencerelevant chemical-shift perturbation, indicating that gly-cyrrhizin does not alter the overall protein structure. Theglobalpreservationof thesecondarystructurewasfurtherconfirmed by circular dichroism (CD) spectroscopy( Figure 3F).Resonances with significant CSD (deviating more thanone standard deviation from the mean CSD) included res-idues F17, Q20 (side-chain protons), R23, E25, K43, andC44 for box A (Figures 3C, 4 A, and 4B) and R109, I112, D123,andA125for boxB (Figures 3C,4C,and 4D).No in- teractions were observed with the linker region betweenthe two boxes and the C terminus. The individual peakscorresponding to the acidic tail residues could not be as-signed because of high spectral overlap and exchangewith the solvent [25]; however, these peaks did not shiftupon addition of glycyrrhizin, excluding their involvementin the binding.Previous structural studies have clearly demonstratedthat the two HMG boxes behave as rigid independent do-mainsthatdonotinteractwitheachotherinthecontextof the full-length protein [25]; we therefore verified whetherglycyrrhizin binds similarly to the two individual HMGboxes (  15 N-labeled box A and box B) ( Figures 3D and3E). Indeed, residues mostly affected by the presence of the ligand all clustered on the first two helices of bothHMG boxes, and coincided with the residues showinghigh CSD in the full-length protein. Additional residues lo-cated on helices 1 and 2 (H26, K42, E107, K113, G122,G129, M133) were more affected in the isolated domainsthan within the entire protein. This effect might be due toa slightly different orientation of the ligand, or to smallstructuraldifferencesoftheentireproteinwhencomparedto the isolated domains.OnbothHMGboxes(whetherinHMGB1orinisolation),thebindingsiteislocatedatthecruxofthetypicalL-shapefold,whichhasa smallsolvent-exposedhydrophobic sur-face suitable for favorable van der Waals interactions withthe triterpene scaffold of glycyrrhizin ( Figure 4 ). Interest-ingly, in the upfield region of the monodimensional protonspectrum of both HMGB1 and box A, variations in chem-ical shifts are also observed for the well-resolved  1 Hmethyl resonances of V19 and V35, located on helices 1and 2 of box A, further suggesting an involvement of thisregion in the binding (see Figure S1 in the Supplemental Data available with this article online). The titration exper-imentsalsoindicate thathelix3isnotinvolved inthebind-ing (Figures 3C–3E and 4 ). Figure 2. Glycyrrhizin Inhibits the Mito-genic Activity of HMGB1 on Vessel- Associated Stem Cells (Mesoangio-blasts) D16mesoangioblastsweregrowninRPMIme-dium containing no additions, 1 nM HMGB1with the indicated concentration of glycyrrhi-zin, or 20% fetal calf serum (FCS). Each pointrepresents the mean of three replicates. Errorbars are omitted to avoid clutter; the standarddeviation was between 2% and 15% of theaverage. The experiment was repeated threetimes. The inhibitory activity of glycyrrhizinon HMGB1-induced stem-cell proliferationis highly significant (p < 0.001 in ANOVA analysis). Chemistry & Biology  14 , 431–441, April 2007 ª 2007 Elsevier Ltd All rights reserved  433 Chemistry & Biology Glycyrrhizin Inhibits HMGB1 Activities  Figure 3. NMR Titration of HMGB1, Box A, and Box B with Glycyrrhizin (A)SuperpositionoftheHSQCspectraofHMGB1without(black)andwith(red)a4-foldexcessofglycyrrhizin.Thespectrawereacquiredat600MHz,T = 298K. Protein concentration was 0.25 mM, in 20 mM phosphate buffer (pH 7.3), 150 mM NaCl, 5 mM DTT.(B)AselectedregionofHMGB1spectraduringthetitrationwithglycyrrhizin(0.5,1,1.5,2,3,4equivalentsofligand).Thestartingandendpointsofthetitration are represented in black and red, respectively. The largest shifts were observed for the amino group of Q20. The observed chemical-shiftchanges are a continuous and monotonic function of the amount of added peptide, indicating that the binding is in the fast exchange limit on theNMR timescale.(C–E)HistogramsshowingthevalueoftheCSDinHMGB1(C),boxA(D),andboxB(E)induceduponbindingwithglycyrrhizin(4-foldexcess).Residuenumbers are indicated on the x axis (residues for which CSD is missing are either prolines or could not be detected because of exchange with thesolvent at pH 7.3); the asterisks indicate the CSD of the amino group of Q20. The continuous line represents the average chemical-shift difference;the dotted line represents the average chemical-shift difference plus one standard deviation. The amino acids mostly affected by the addition of gly-cyrrhizin are located on the first two helices of box A and box B (helices are schematically represented at the top of the histograms).(F)CDspectraofHMGB1(3 m M)without(continuousline)andwith(dottedline)200 m Mglycyrrhizin.Thespectrashowthatuponadditionofanexcessof ligand, the secondary structure of the protein does not change. Spectra were acquired at 20  C in 10 mM phosphate buffer (pH 7.3), 10 mM NaCl. 434  Chemistry & Biology  14 , 431–441, April 2007 ª 2007 Elsevier Ltd All rights reserved Chemistry & Biology Glycyrrhizin Inhibits HMGB1 Activities  Werepeatedthetitrationswithcarbenoxolone,aderiva-tive of glycyrrhizin where the dimer of glucuronic acid issubstituted by a succinyl moiety ( Figure 1 A). In the caseof box A, we found that the highest chemical-shift pertur-bationsofthebackbone 1 Hand 15 Nwereagainlocatedonthe first two helices, as observed for glycyrrhizin (data notshown).However,thespectraforboxBandthefull-lengthprotein at higher carbenoxolone concentrations were of low quality, and showed evidence of aggregation. Modeling of Glycyrrhizin Interaction with Box A  To further characterize the interaction between glycyrrhi-zin and the HMG boxes, we ran classical X-filtered exper-iments on the box A:glycyrrhizin complex. Because of themodest affinity of glycyrrhizin (see below), we observedvery few unambiguous intermolecular interactions, whichwerenot sufficienttocalculatea structureof thecomplex.We therefore adopted the HADDOCK strategy [26], arecent but well-established procedure which uses Figure 4. Mapping of the Glycyrrhizin Binding Sites and Model of Interaction (A) Ribbon and (B) surface representation of box A (PDB code: 1aab ); the side chains of residues in the full-length protein showing CSD larger than the mean value plus one standard deviation (F17, Q20, R23, E25, K43, C44) are shown in red.(C) Ribbon and (D) surface representation of box B (PDB code: 1hmf  ); the side chains of residues in the full-length protein showing CSD larger thanthe mean value plus one standard deviation (R109, I112, D123, A125) are shown in red.(E) Model of interaction of glycyrrhizin with box A; HADDOCK restraints and hydrophobic residues are shown in red and magenta, respectively.Glycyrrhizin (cyan) accommodates at the junction of the two arms of the L-shape fold.(F)Zoomofthebinding site:glycyrrhizinestablishes favorablevanderWaalsinteractionswiththehydrophobic sidechainsofY15,F37,A16,andV19(magenta).ThecomplexisfurtherstabilizedbyabifurcatedelectrostaticinteractionbetweenthecarboxylateofglycyrrhizinandtheamineofK42andthe hydroxyl group of Y15 (yellow dashed lines). The carbonyl group of glycyrrhizin forms a stable hydrogen bond with the guanidinium group of R23(red dashed line). Chemistry & Biology  14 , 431–441, April 2007 ª 2007 Elsevier Ltd All rights reserved  435 Chemistry & Biology Glycyrrhizin Inhibits HMGB1 Activities
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